This week, a major problem has been detected. The primers used originally had the wrong sequence, so they had to be redesigned and reordered. We found out that the insert was not in the LR product or the BP product, so even if everything worked, it does not contain my insert of interest. So, back to the drawing board!
When the correct primers arrived at the lab, I decided to run both Gateway and Traditional cloning concurrently, to see which method worked better. I ran PCRs of the insert for both cloning methods, and then ran gels for both to ensure that the insert was in the PCR product. Then, for Gateway cloning, everything will be done the same way as discussed previously. For traditional cloning, DNA ligase will be used to put the insert into the PET vector.
I spent the past couple of days using restriction enzymes to cut the vector and the PCR product to get them ready for insertion, and ran more PCRs and gels for BP clonase colonies to make sure that the insert is in the vector, and that the transformation was successful.
Once transformation has been performed by E coli, the final product will be isolated and expressed. This mistake taught me that starting over again does not mean the end of the world. Having put one month of hard work behind me, I am ready to use all of the techniques and methods I learned to put my best foot forward for the rest of my time here.